Electroporation of cDNA/Morpholinos to targeted areas of embryonic CNS in Xenopus.

BACKGROUND: Blastomere injection of mRNA or antisense oligonucleotides has proven effective in analyzing early gene function in Xenopus. However, functional analysis of genes involved in neuronal differentiation and axon pathfinding by this method is often hampered by earlier function of these genes during development. Therefore, fine spatio-temporal control of over-expression or knock-down approaches is required to specifically address the role of a given gene in these processes. RESULTS: We describe here an electroporation procedure that can be used with high efficiency and low toxicity for targeting DNA and antisense morpholino oligonucleotides (MOs) into spatially restricted regions of the Xenopus CNS at a critical time-window of development (22-50 hour post-fertilization) when axonal tracts are first forming. The approach relies on the design of "electroporation chambers" that enable reproducible positioning of fixed-spaced electrodes coupled with accurate DNA/MO injection. Simple adjustments can be made to the electroporation chamber to suit the shape of different aged embryos and to alter the size and location of the targeted region. This procedure can be used to electroporate separate regions of the CNS in the same embryo allowing separate manipulation of growing axons and their intermediate and final targets in the brain. CONCLUSION: Our study demonstrates that electroporation can be used as a versatile tool to investigate molecular pathways involved in axon extension during Xenopus embryogenesis. Electroporation enables gain or loss of function studies to be performed with easy monitoring of electroporated cells. Double-targeted transfection provides a unique opportunity to monitor axon-target interaction in vivo. Finally, electroporated embryos represent a valuable source of MO-loaded or DNA transfected cells for in vitro analysis. The technique has broad applications as it can be tailored easily to other developing organ systems and to other organisms by making simple adjustments to the electroporation chamber.

Protein repellent properties of covalently attached PEG coatings on nanostructured SiO(2)-based interfaces.

In this study, we report the systematic comparison of different poly(ethylene glycol) (PEG) self-assembled monolayers on glass with respect to their protein adsorption and cell adhesion resistance. Combining PEGylation with micellar nanolithography allowed the formation of gold nanoparticle arrays on glass and selective coverage of the free glass area by PEG. The gold nanoparticles serve as anchor points for the attachment of individual proteins and peptides such as the cell-matrix adhesion promoting cyclic RGDfK motif or the kinesin motor protein Eg5. The capability of the motor protein to bind microtubules remained unaffected by the immobilization. It was shown that the film thickness of a water swollen PEG layer is crucial to maximize the interaction between proteins and peptides with the nanostructures. Non-specific interaction between cells or microtubules and the surface was minimized. The optimum PEG layer thickness correlated with the size of gold nanoparticles which was approximately 5 nm.

E3 ligase Nedd4 promotes axon branching by downregulating PTEN.

Regulated protein degradation via the ubiquitin-proteasome system (UPS) plays a central role in building synaptic connections, yet little is known about either which specific UPS components are involved or UPS targets in neurons. We report that inhibiting the UPS in developing Xenopus retinal ganglion cells (RGCs) with a dominant-negative ubiquitin mutant decreases terminal branching in the tectum but does not affect long-range navigation to the tectum. We identify Nedd4 as a prominently expressed E3 ligase in RGC axon growth cones and show that disrupting its function severely inhibits terminal branching. We further demonstrate that PTEN, a negative regulator of the PI3K pathway, is a key downstream target of Nedd4: not only does Nedd4 regulate PTEN levels in RGC growth cones, but also, the decrease of PTEN rescues the branching defect caused by Nedd4 inhibition. Together our data suggest that Nedd4-regulated PTEN is a key regulator of terminal arborization in vivo.

Phosphorylation by Cdk1 increases the binding of Eg5 to microtubules in vitro and in Xenopus egg extract spindles.

BACKGROUND: Motor proteins from the kinesin-5 subfamily play an essential role in spindle assembly during cell division of most organisms. These motors crosslink and slide microtubules in the spindle. Kinesin-5 motors are phosphorylated at a conserved site by Cyclin-dependent kinase 1 (Cdk1) during mitosis. Xenopus laevis kinesin-5 has also been reported to be phosphorylated by Aurora A in vitro. METHODOLOGY/PRINCIPAL FINDINGS: We investigate here the effect of these phosphorylations on kinesin-5 from Xenopus laevis, called Eg5. We find that phosphorylation at threonine 937 in the C-terminal tail of Eg5 by Cdk1 does not affect the velocity of Eg5, but strongly increases its binding to microtubules assembled in buffer. Likewise, this phosphorylation promotes binding of Eg5 to microtubules in Xenopus egg extract spindles. This enhancement of binding elevates the amount of Eg5 in spindles above a critical level required for bipolar spindle formation. We find furthermore that phosphorylation of Xenopus laevis Eg5 by Aurora A at serine 543 in the stalk is not required for spindle formation. CONCLUSIONS/SIGNIFICANCE: These results show that phosphorylation of Eg5 by Cdk1 has a direct effect on the interaction of this motor with microtubules. In egg extract, phosphorylation of Eg5 by Cdk1 ensures that the amount of Eg5 in the spindle is above a level that is required for spindle formation. This enhanced targeting to the spindle appears therefore to be, at least in part, a direct consequence of the enhanced binding of Eg5 to microtubules upon phosphorylation by Cdk1. These findings advance our understanding of the regulation of this essential mitotic motor protein.

Spatial and temporal control of mitotic cyclins by the Gnu regulator of embryonic mitosis in Drosophila.

Mutation of the Drosophila maternal cell cycle regulator, Gnu, results in loss of embryonic mitosis and the onset of excessive nuclear DNA replication. The Gnu phosphoprotein is normally synthesized in nurse cells and transported to the developing oocyte. We created a gnuGFP-bcd3'UTR transgene using the gnu promoter and bicoid 3'UTR, that translates GnuGFP only on egg activation from a localized anterior source. This transgene was able to rescue the sterility of gnu mutant females. Gnu is therefore first required after egg activation for polar body condensation and zygotic mitoses. Embryos containing pronounced anterior-posterior gradients of Gnu activity demonstrate that Gnu regulates mitotic activity by promoting cyclin B stability. Our gnuGFP-bcd3'UTR vector provides a novel experimental strategy to analyse the temporal requirement and role of cell cycle regulators including potential sperm-supplied factors in eggs and embryos.